In the present work the hypericin interaction with hemoglobin was studied by absorption and fluorescence spectroscopy both under incubation in dark and visible light exposure. An absorption reduction in Soret band of hemoglobin (407 nm) was revealed under the photodynamic influence and incubation in dark with hypericin that had hypericin concentration and time dependent manner. Hypericin reduced the intensity of the hemoglobin emission peaks at 334 and 421 nm, correlating with hypericin concentration, incubation and irradiation time. An obvious increase in electrophoretic mobility of hemoglobin was observed under the incubation with hypericin. Simultaneously, a partial conversion of hemoglobin to met-hemoglobin and a pH decrease in hemoglobin solution were detected. Structural changes of hemoglobin caused by hypericin were accompanied by a change in peroxidase activity of the protein. Thus under the hypericin influence hemoglobin properties as a hydrogen peroxide detector could be improved and an effective determination of peroxide formation could be achieved. This makes hemoglobin an attractive ‘recognition’ element for construction of third-generation biosensors.